Helicobacter pylori utilises urea for amino acid synthesis

IMMUNOLOGY AND MEDICAL MICROBtOLOGY

ELSEVIER

FEMS Immunology

and Medical Microbiology

I3 ( 1996) 87-94

Helicobacter pylori utilises urea for amino acid synthesis C.L. Williams

a**, T. Preston ‘, M. Hossack b, C. Slater ‘, K.E.L. McCall b

a Department of Microbiolog_v, Royal Alexandra Hospital, Paisle! PA2 YPN. UK h Department of Medicine and Theraputics. University of Glasgow. Glasgow, UK ’ S.U.R.R.C.. East Kilbride. UK Received 29 June 1995: revised 2 1 September

1995; accepted 2 1 September

1995

Abstract Helicohacfer p?;/ori has one of the highest urease activities of all known bacteria. Its enzymatic production of ammonia protects the organism from acid damage by gastric juice. The possibility that the urease activity allows the bacterium to utilise urea as a nitrogen source for the synthesis of amino acids was investigated. H. pylori (NCTC 116381 was incubated with 50 mM urea, enriched to 5 atom% excess 15N, that is the excess enrichment of 15N above the normal background, in the presence of either NaCl pH 6.0, or 0.2M citrate pH 6.0. E. coli (NCTC 90011 was used as a urease-negative control. 15N enrichment was detected by isotope ratio mass spectrometry. H. pyfori showed intracellular incorporation of “N in the presence of citrate buffer pH 6.0 but there was no significant incorporation of 15N in unbuffered saline or by E. coli in either pH 6.0 citrate buffer or unbuffered saline. The intracellular fate of the urea-nitrogen was determined by means of gas chromatography/mass spectrometry following incubation with 15N enriched 5 mM urea in the presence of either 0.2 M citrate buffer pH 6.0 or 0.2 M acetate buffer pH 6.0. After 5 min incubation in either buffer the 15N label appeared in glutamate, glutamine, phenylalanine, aspartate and alanine. It appears, therefore, that at pH and urea concentrations typical of the gastric mucosal surface, H. pylori utilises exogenous urea as a nitrogen source for amino acid synthesis. The ammonia produced by H. pylori urease activity thus facilitates the organism’s nitrogen metabolism at neutral pH as well as protecting it from acid damage at low pH. Keywords: Hrlicobacter pylori; Urease: Amino acid; Mass spectrometry

1. Introduction Helicobacter pylori infection is present in approximately 40% of the world’s population [I]. The infection is now recognised as the most important acquired factor in the development of duodenal ulcer disease [2] and has been shown to be associated with

* Corresponding

author.

Federation of European Microbiological SSDf 0928-8244(95)00088-7

Societies.

the development of gastric carcinoma [3]. Unfortunately the infection is difficult to eradicate with currently available antibiotics [4]. New approaches to eradication therapy may depend upon a fuller understanding of the metabolism of this common bacterium. H. pylori is remarkable because of its high urease enzyme activity. The rate of urea hydrolysis by the organism is at least ten times as great as that of Proteus mirabilis. The enzyme has a high affinity for

88

CL. Williams et al. / FEMS Immunology md Medical Microbiolo~y~ 13 (I YY6) 87-W

urea with a Km of 0.48 mM [5] and has a V,,,,, of 1100 k 200 nmol urea min-’ mgg ’ [6]. Electron microscope studies showed that the urease enzyme was situated on the outer membrane of the bacterium [7,8] and it has been postulated that the ammonia produced by urea hydrolysis remains outside the bacterium to protect the organism from the acidic, gastric environment. However, in earlier studies it was observed that all of the ammonia expected from the hydrolysis of urea could not be recovered from the incubate [9]. The amount of ammonia recovered was related to the extracellular concentration of urea, ranging from 27% recovery at 5 mM urea to 3% at 50 mM urea. These studies also showed that the survival of H. pylori was decreased following 5 min incubation in 0.2 M citrate buffer at pH 6.0 containing 50 mM urea: under these conditions only 9% of the bacteria survived [ 101. This effect seems to be specific for Helicobacter species and does not occur with other urease-producing bacteria [ 111. Normally bacterial cells growing in an excess of ammonia detoxify it by the reductive amination of cY-ketoglutarate with the subsequent formation of glutamate. Ammonia is also used to convert a portion of the glutamate to glutamine in an energy-requiring reaction catalysed by glutamine synthetase [ 121. In the case of H. pylori, it may be that the presence of 0.2 M citrate and 50 mM urea at pH 6.0 causes an imbalance between the formation of cY-ketoglutarate by the tricarboxylic acid (TCA) cycle and its consumption during the formation of glutamate and glutamine. Under the conditions described, citrate may buffer the intracellular pH to 6. At this pH, 98% of the ammonia will be protonated and thus be unable to diffuse back out of the cell [ 131. This increased concentration of intracellular nitrogen may result in the depletion of the essential metabolite cY-ketoglutarate with consequent cessation of energy production and cell death. There is some evidence to support this hypothesis. When H. pylori is pre-incubated with a-ketoglutarate prior to exposure to 0.2 M citrate and 50 mM urea at pH 6.0, then the survival of, the organism is increased from 9% to 45% [14]. If bacterial cell death is due to ar-ketoglutarate depletion, then nitrogen derived from extracellular urea should be detectable within the bacterial cell in the form of glutamate and, as glutamate is an impor-

tant substrate for intracellular transamination reactions, urea-derived nitrogen should also be detectable in a wide variety of amino acids. To test this hypothesis, studies by both isotope ratio mass spectrometry (IRMS) and gas chromatography-mass spectrometry (GC-MS ) with “N urea tracer, firstly. to detect the presence of urea derived nitrogen within the bacterial cell and, secondly, to determine its metabolic fate, are described here. In our earlier studies the recovery of nitrogen from the incubate was reduced in the presence of both 0.2 M citrate and 0.2 M acetate buffers, but survival was only decreased with citrate buffer [9]. Citrate and acetate buffers were therefore used to increase the amount of label likely to be incorporated into the bacterium and thus increase the probability of detecting label within bacterial amino acids.

2. Materials and methods

2.1. Studies

qf

urea nitrogen

incorporation

into the

bacteriul cell by IRMS H. pylori (NCTC 11638) was inoculated onto blood agar plates containing Skirrow’s selective supplement (Oxoid) and 1% (w/v) amphotericin (Squibb) and incubated for 72 h at 37°C in a microaerophilic atmosphere (BBL Campypak). Growth from the plate was suspended in 0.9% (w/v) NaCl, in a sterile universal container to McFarland standard 10 and placed in a water-bath at 37°C. A 20 X 8 mm tin capsule (Elemental Micronalysis, Okehampton, Devon, UK) with a capacity of 600 ~1 was placed inside a l-ml plastic Eppendorf tube. The tube was placed in a water-bath at 37°C and allowed to equilibrate. The bacterial suspension (300 ~1) was pipetted into the tin capsule, and 300 ~1 of one of the following was added to the bacterial suspension: (a) 0.9% (w/v) unbuffered NaCl, pH 6.0, with 50 mM unenriched urea; (b) 0.9% (w/v) unbuffered NaCl, pH 6.0, with 50 mM urea enriched with 5 atom% “N urea [ 151; or (c) 0.2 M citrate, pH 6.0, with 50 mM urea enriched with 5 atom% “N urea. The

C.L. Williums

et al. / FEMS Immunology

biologically active D isomer of citrate was used throughout. All buffers were placed in a water-bath at 37°C and allowed to equilibrate before use. The tubes were incubated in the water-bath at 37°C for either 1 or 5 min. Following this incubation. the bacteria1 suspensions were centrifuged at 6000 rpm (2000 X g) for 5 min in a microcentrifuge at room temperature. The bacteria1 pellet was resuspended in 600 ~1 of 0.9% (w/v) NaCl at pH 6.0 and washed twice to remove any “N label which may have adsorbed onto the surface of the bacteria. In addition to the H. pylori. a urease-negative control (E. coli NCTC 9001) was used to ensure that the washing procedure was sufficient to remove all of the tracer which was not incorporated into the bacteria. After washing. the bacteria were present as pellets at the bottom of the tin capsules. The tin capsules were removed from the Eppendorf tubes and the pellets were freeze-dried overnight. The lower end of the capsule containing the bacterial pellet was cut off and folded until it was small enough to fit into the autosampler of the elemental analyser connected on line to the isotope ratio mass spectrometer. The scissors were thoroughly cleaned between each use. Nitrogen standards (50 and 100 pg) were prepared by drying 50 and 100 ~1 respectively of a 1 mg N ml-’ solution of ammonium sulphate (BDH Chemicals) into tin capsules. All samples were then analysed on a Roboprep elemental analyser (Europa Scientific) on line with a MM602 isotope ratio mass spectrometer (Fisons Instruments). The results were analysed using proprietary software (Europa Scientific) running on an IBM compatible PC and were expressed in atom% 15N [IS] 2.2. Studies ofthe fate of the urea nitrogen incorporated h,ithin the bacterial cell using GC-MS A McFarland standard 10 suspension of H. pylori (5.0 ml) was prepared as described earlier. 2.0 ml of this suspension was added to 2.0 ml of one of the following buffers: (i) 5 mM 99 atom% excess “N urea [ 151 in 0.2 M citrate buffer, pH 6.0; (ii) 5 mM 99 atom% excess 15N urea in 0.2 M acetate buffer, pH 6.0.

and Mediccrl Microbiology

13 ClYY61 87-Y4

89

The buffers were equilibrated at 37°C before the start of the experiment. The suspension was incubated at 37°C in a water-bath for 5 min and 3.0 ml aliquots taken. One ml was sonicated until all bacterial cells were shown to be disrupted as confirmed by Gram staining. The remaining 2 ml of the suspension was filtered through a 0.22 pm filter and the filtrate collected. Following filtration or sonication. samples were rapidly frozen and stored at - 20°C. In preparation for cation exchange purification prior to CC-MS analysis, 1.O ml of each sample was added to 9.0 ml of de-ionised distilled water (DDW) and cycloleucine (250 nmol) was added as an internal standard. Fifty ~1 of 1.2 M HCI was added to adjust the pH to 2-3. The samples were loaded onto a AGW 50-X8 (H + ) cation exchange column (Polyprep: Biorad), which had been washed with 20 ml of DDW. until no further colour was eluted. and washed again with 10 ml DDW. The amino acid fraction was eluted from the column with 5.0 ml of 2 M NH,OH followed by 5.0 ml of DDW. The eluted fraction was dried in a vacuum centrifuge, reconstituted in 400 ~1 DDW and transferred to a 2.0 ml derivatization vial. Sarcosine (250 nmol) was added as an internal standard. Amino acid tert-butyldimethylsilyl (TBDMS) derivatives were formed following addition of 25 ~1 acetonitrile and 25 ~1 N-trrt-butyIdimethylsilyl-Nmethylflouroacetamide (Regis) and heating at 60°C for I h [ 161. The GC column was a high resolution bonded phase capillary column (30 m X 0.25 mm DB-SMS. 0.25 pm film: J&W Scientific, USA). The temperature programme was: I lO”C-2 10°C at 10”C/min: 2lO”C-310°C at 20”C/min; hold for I min. The MS was a VG Trio-1000 (Fisons Instruments, UK). The electron impact ion source was operated at 150 JLA trap current and 45 eV electron energy. The samples were first analysed in scanning mode to confirm the presence of individual amino acids. and to investigate other potential components. The “N enrichment of each of the larger components (in this case urea and glutamine) was then analysed in selected ion recording (SIR) mode. Finally, the 15N enrichment of other amino acids was analysed in SIR mode, at high sensitivity for improved precision.

90

C.L. Williams et al./FEMS

Immunology and Medical Microbiology

2.3. Statistics

Results were compared by an unpaired Student’s t-test.

13 (19%) 87-94

min incubations when compared to 0.9% (w/v) saline with 50 mM urea (P(O.001). Again in the presence of 0.2 M citrate, there was more intracellular “N after 5 min incubation than after 1 min (P(O.05). These results are summarised in Fig. 1.

3. Results 3.2. Studies of the fate of the urea-nitrogen 3.1. Studies of urea-nitrogen bacterial cell using IRMS

incorporation

into the

These experiments measured the effect of citrate buffer on the incorporation of “N urea tracer into the bacterial cell and were performed on six occasions in triplicate. Results are expressed as atom% excess I5N. There was significant intracellular incorporation of 15N following a 5 min incubation with 0.9% (w/v> saline with 50 mM urea when compared to the unen~ched 0.9% (w/v) saline control (background) (P(O.05). There was no significant “N incorporation after 1 min incubation. In the presence of 0.2 M citrate, the incorporation of 15N was markedly increased after both 1 and 5

rated within the bacterial cell following with citrate bu@er using GOMS

incorpoincubation

When H. pylori was incubated for 5 min at 37°C in either 0.2 M citrate or 0.2 M acetate buffers with 5 mM 15N urea, 15N enrichment was found in a number of amino acids isolated from a cell sonicate. Figs. 2 and 3 show the total ion chromatograms from sonicates of H. pylori following incubation in citrate and acetate. These were obtained by summing the results of each channel of a GC-MS analysis in selected ion monito~ng mode. The 15N enrichment of each amino acid was calculated by comparing the peak area ratios of appropriate fragment ions with those of an unenriched standard (the data were ex-

n

background

** = pI

Acetate buffer

r

11 7

fnternal Standard

5

)

Ph

)hate

x3

Urea l ‘**

.h2’ Pha ‘***

Gin*

Internal Standard 8

:FS

16

Ala l **

34 I

16.23,

Asp *** Gfy l *

Pro l *

Fig. 3. Total ion chromatogram fmm sonicates of ff. p&xi following incubation in 0.2 M acetate with 5 mM “N urea at pH 6.0. These were obtained by summing the results of each channel of a GC/MS analysis in selected ion monitoring mode. The “N enrichment of each amino acid was calculated by comparing the peak area ratios of appropriate fragment ions with those of an unenriched standard (the data rsN, /$PE ‘TN). * * * * were expressed in atom% excess = 50-99 atom% excess “N. ” * * = 20-49 atom% excess “N. * ’ = S- 19 atom%, excess “N. ’ = 0.2-4.9 atom% excess ‘“N.

Table 1 Table showing incorporation of urea derived nitrogen into bacterial amino acids following 5 min incubation in 0.2 M citrate and acetate buffers at pH 6.0 with 5 mM ‘“N urea. The “N enrichment of each amino acid was calculated by comparing the peak area ratios of appropriate fragment ions with those of an unenriched standard (the data were expressed in atom% excess “N. APE “N) Treatment

Gln

Glu

Phe

Asp

Ala

Gly

Pro

Ser

Citrate buffer Acetate buffer

0.45 0.45

28 64

24 75

15 34

15 29